Project Abstract This is an application for renewal of a grant to study picornavirus genome replication. Picornaviruses represent an existing and emerging threat to US public health. Although protein factors and genetic elements required for picornavirus genome replication are known and appear to be conserved, a clear understanding of the mechanisms employed to produce picornaviral RNA is lacking. The long-term objective of this program is to reconstitute picornavirus genome replication in vitro from purified components. We have achieved all of the major objectives of the previous funding period. In addition, we have solved the first crystal structure for a picornaviral 3CD protein, developed the technology to study 3C-RNA interactions by using nuclear magnetic resonance spectroscopy, and discovered that the 3CD protein has both pre- and post-replication functions. During the next funding period, we will continue our studies of picornavirus genome replication as well as explore our newly discovered function for 3CD by pursuing the following specific aims: (1) Define the mechanism of assembly and structural organization of the picornavirus VPg uridylylation complex by using molecular genetic, biochemical and biophysical approaches; (2) Define the molecular basis for sequence- and structure-specific RNA recognition by 3C by using nuclear magnetic resonance spectroscopy; and (3) Elucidate the function of 3CD in formation of replication complexes.